T cell proliferation in-vitro culture method and application thereof
By combining CD3e antibody with β-mercaptoethanol and adjusting the composition and concentration of the culture medium, the problem of controlling cell activity and proliferation in T cell in vitro culture was solved, achieving precise regulation of T cell proliferation and meeting the needs of drug efficacy verification.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG UNIV OF TECH
- Filing Date
- 2024-03-04
- Publication Date
- 2026-04-10
AI Technical Summary
In existing technologies, it is difficult to control the cell activity and proliferation of T cells during in vitro culture, which makes it difficult to verify the efficacy of drugs, especially to distinguish between the promoting or inhibiting effects of drugs.
By combining CD3e antibody with β-mercaptoethanol and adjusting their concentration ratio and culture medium composition, the basal proliferation of T cells can be controlled. An RPMI 1640 culture medium containing 15% fetal bovine serum, 1% penicillin and streptomycin, 20 mM HEPES, 5 ng/mL IL-2 and different concentrations of β-mercaptoethanol is provided, simplifying the culture medium composition to a cheap and readily available one, relying solely on CD3 to provide activation signals.
Maintaining cell viability at around 30% within three days enables precise control of T cell proliferation, with proliferation regulation ranging from 5% to 80%, meeting the validation needs of different functional drugs and improving the visualization of drug efficacy.
Smart Images
Figure CN121825872A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of immunological drug development. Specifically, it relates to a method for culturing T cell proliferation in vitro and its application. BACKGROUND
[0002] In the process of drug development for immune-related diseases, there are different needs. For example, for autoimmune diseases, it is necessary to inhibit the excessive proliferation of immune cells and excessive effector function, and to develop immunosuppressive drugs, at which time a model of excessive activation of immune cells needs to be established. For cancer, it is necessary to proliferate immune cells at the target site and enhance their effector function to achieve the purpose of treatment, and to develop immune-enhancing drugs, at which time a model of slight activation of immune cells needs to be established and the activity of the cells in vitro needs to be maintained as much as possible. Therefore, the establishment of different models in vitro is crucial for the development of drugs for immune-related diseases.
[0003] Lymphocyte proliferation test is an important indicator for measuring the promotion or inhibition of lymphocyte proliferation by drugs. Mouse spleen lymphocytes are isolated, stained with CFSE (carboxyfluorescein diacetate succinimidyl ester), and the cells obtain a fluorescence signal that decreases with the degree of division. The proliferation peak can be detected by flow cytometry. According to the change of CFSE proliferation peak relative to the background proliferation peak after drug addition, the effect of the drug on T cells can be judged. To make T cells proliferate in vitro, T cells must be activated, which is also the key step of this experiment. T cell activation requires "first signal" CD3 signal and "second signal" CD28 costimulation, but only T cells must receive two signals to proliferate. Effector T lymphocytes and memory T lymphocytes in the spleen receive the "first signal" of T lymphocyte activation given by anti-CD3e antibody, and can proliferate.
[0004] Traditional experimental scheme: add 50 μl of 5 μg / ml anti-CD3e antibody solution to each well of a 96-well plate, incubate in a 37°C incubator for 2h or 4°C overnight. Prepare a single cell suspension of mouse spleen lymphocytes, stain with 5 μM CFSE, incubate in the dark at room temperature for 15 minutes, add 5 ml of complete culture medium to release unbound CFSE, incubate in the dark at room temperature for 5 minutes, wash twice with culture medium, centrifuge and resuspend the cells in new complete culture medium to 3×10 6 Remove the CD3 antibody solution from the 96-well plate, add 200 μl of cell suspension to each well. Add anti-CD28 5 μg / mL and IL-2 20 ng / mL to the incubator for 3-4 days.
[0005] In the traditional experimental scheme, only 10% fetal bovine serum, 1% penicillin and streptomycin and 10 ng / ml IL-2 RPMI 1640 medium are used as T cell culture medium, and the "first signal" CD3 signal and "second signal" CD28 are used for T cell activation. In this scheme, the activity of the cells is difficult to guarantee, the proliferation of the cells is difficult to control, and the CFSE cell proliferation curve that can be used for functional analysis of immunotherapy drugs is difficult to obtain, which is a difficulty in visualizing the efficacy of immunotherapy drugs. In other T cell culture schemes, most of them are for T cell immunotherapy, and by adding components, the activity and proliferation ability of T cells in vitro are improved.
[0006] In summary, at present, the survival rate of T cells is low and cannot be stably survived; the proliferation of T cells is difficult to control, for example, when verifying the promotion ability of drugs on T cell proliferation, if the experimental scheme leads to too high background proliferation, the promotion effect of the drug cannot be seen; similarly, when verifying the inhibition ability of drugs on T cell proliferation, if the experimental scheme leads to too low background proliferation, the inhibition effect of the drug cannot be seen; the concentration of anti-CD3e antibody is adjusted alone to adjust the background proliferation degree of T cells, but the influence of its concentration on cell proliferation is large and difficult to control, and it is easy to lead to cell activation failure and death. SUMMARY
[0007] In view of the problems existing in the present T cell proliferation, the present application provides a method for in vitro proliferation culture of T cells, which introduces β-mercaptoethanol to assist CD3e antibody to adjust the cell proliferation degree, so that better control effect can be achieved. A kit prepared from the related raw materials of the method is provided for detecting the activity of immunological preparations.
[0008] The scheme of the present application is as follows:
[0009] A method for in vitro culture of T cell proliferation, CD3e antibody and β-mercaptoethanol are used to control the background proliferation of T cells.
[0010] Preferably, the mass ratio of the added amount of CD3e antibody and β-mercaptoethanol is 0.04-0.8.
[0011] Preferably, it specifically comprises the following steps:
[0012] (1) CD3e antibody is coated with antibody;
[0013] (2) Prepare a lymphocyte single cell suspension and dye;
[0014] (3) Resuspend the cell suspension of step (2) with a culture medium containing β-mercaptoethanol;
[0015] (4) After removing the solution in step (1), add the cell suspension from step (3), incubate, and then perform flow cytometry detection.
[0016] Furthermore, the process of step (1) is as follows: the anti-CD3e antibody is incubated in a 37°C incubator for 2 hours to coat the antibody.
[0017] Furthermore, the process of step (2) is as follows:
[0018] S1. A single-cell suspension of lymphocytes was stained with CFSE, incubated at room temperature in the dark, and then added with complete culture medium to release unbound CFSE. The suspension was then incubated at room temperature in the dark and washed with complete culture medium.
[0019] S2 cells were resuspended in a medium containing 15% fetal bovine serum, 1% penicillin-streptomycin, 20 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 5 ng / mL IL-2, and 13.75 μM–55 μM β-mercaptoethanol to a concentration of 3–5 × 10⁻⁶ cells / mL. 6 per mL.
[0020] Furthermore, step (3) involves removing the anti-CD3e antibody solution coated in step (1), adding the cell suspension from step (2), incubating in a 37°C incubator for three days, and then detecting the cells using flow cytometry.
[0021] Furthermore, the fetal bovine serum in step S1 needs to be heat-inactivated; and / or,
[0022] The culture medium needs to be filtered.
[0023] Furthermore, the conditions for heat inactivation are 50-60℃ for 10-50 minutes.
[0024] The above method is applied to the in vitro culture of T cell proliferation; preferably, the application is used to detect the activity of immunomodulatory drugs.
[0025] Another object of the present invention is to protect a kit for detecting the activity of an immunoassay agent, prepared using the raw materials described above.
[0026] Preferably, the immunomodulator is an immunosuppressant and / or an immunoactivator.
[0027] When detecting the immunosuppression effect of immunosuppressive drugs, the scheme of this invention can be used with high concentrations of anti-CD3e antibody coating and high concentrations of β-mercaptoethanol to synergistically enhance the basal proliferation effect. When detecting immunostimulating drugs, low concentrations of anti-CD3e antibody should be used, as this may lead to insufficient activation signals received by T cells, resulting in cell death. On the other hand, adding high concentrations of β-mercaptoethanol at this time may induce apoptosis. In this case, an appropriate concentration of β-mercaptoethanol should be added to maintain the cell's viability. When the inhibitory or stimulating function of an unknown drug is known, the concentrations of CD3 antibody and β-mercaptoethanol can be adjusted to keep the basal proliferation at an intermediate level, thereby making the drug's activation or inhibition effects easier to observe.
[0028] The beneficial effects of this invention are:
[0029] a. A novel T-cell culture medium, namely RPMI 1640 medium containing 15% fetal bovine serum (heat-inactivated at 56°C for 30 min), 1% penicillin and streptomycin, 20 mM HEPES, 5 ng / ml IL-2, and 13.75 μM, 27.5 μM, or 55 μM β-mercaptoethanol. This medium does not require complex components such as growth factors, insulin, transferrin, essential amino acids, and non-essential amino acids. Based on relatively inexpensive and readily available components, it maintains cell viability at approximately 30% within three days, ensuring the desired cell viability for experiments.
[0030] b. Using the T cell culture medium mentioned in this invention, the effect of more precise control of the basal proliferation of T cells can be achieved by changing the coating amount of anti-CD3e antibody and the combination of different concentrations of β-mercaptoethanol in the T cell culture medium mentioned in this invention;
[0031] c. Due to the large number of effector T cells and memory T cells present in the spleen, they can be activated solely by the "first signal" provided by CD3. Unlike traditional methods, this invention does not require the "second signal" provided by CD28; it achieves excellent activation results solely by the "first signal" provided by CD3. Attached Figure Description
[0032] Figure 1 The image shows the CFSE lymphocyte proliferation curve. By adjusting the concentrations of anti-CD3e antibody and β-mercaptoethanol in the protocol, the cell viability rate can be maintained at around 30% for 3 days, while simultaneously regulating the basal proliferation of T cells from below 5% to over 80%.
[0033] Figure 2 This is a diagram showing the effect of the scheme in Example 1 on the high and low regulation of T cell basal proliferation, thus meeting the needs of verifying different functional drugs.
[0034] Figure 3 The cell activity results are for the scheme in Example 1.
[0035] Figure 4 The image shows the proliferation peak effect obtained by using this method to detect a test drug in Example 2.
[0036] Figure 5 This is a graph showing the results of drug-induced lymphocyte proliferation detection in Example 2. Compared with the control group, the immune cell proliferation rate in the group with the added drug increased by more than 15%, indicating that the drug has an immune-promoting effect. Detailed Implementation
[0037] Use reagent catalog
[0038] Fetal bovine serum: Noverse NFBS-2500A
[0039] Penicillin and Streptomycin: Biosharp BL505AHEPES: Sangon Biotech A600264
[0040] Mouse IL-2 recombinant protein: Tonbo Biosciences 21-8021-U005 β-Mercaptoethanol: GIBCO21985023
[0041] RPMI 1640 medium: Gibco C11875500BT; Anti-Mouse CD3e antibody: Tonbo Biosciences 40-0031-U500CFSE; TargetMol T6802
[0042] Example 1: Expansion of mouse spleen lymphocytes
[0043] (1) Antibody coating was performed by incubating 50 μl of 0.625 μg / ml, 1.25 μg / ml, 2.5 μg / ml, or 5 μg / ml anti-CD3e antibody in a 37°C incubator for 2 h;
[0044] (2) Prepare a single-cell suspension of mouse spleen lymphocytes, stain with 5 μM CFSE, incubate at room temperature in the dark for 15 minutes, add 5 ml of complete culture medium to release unbound CFSE, incubate at room temperature in the dark for 5 minutes, and wash twice with complete culture medium.
[0045] (3) Resuspend cells to 5 × 10⁶ cells in RPMI 1640 medium (filtered through a 0.22 μm filter) containing 15% fetal bovine serum (heat-inactivated at 56°C for 30 min), 1% penicillin-streptomycin, 20 mM HEPES, 5 ng / ml IL-2, and 13.75 μM, 27.5 μM, or 55 μM β-mercaptoethanol. 6Cells / mL. Incubate in an incubator for 3 days, then perform flow cytometry analysis.
[0046] from Figure 1 It can be seen that by adjusting the concentrations of anti-CD3e antibody and β-mercaptoethanol in the regimen, the cell survival rate can be maintained at around 30% within 3 days, while the effect of regulating the basic proliferation of T cells can be increased from less than 5% to more than 80%.
[0047] from Figure 2 It can be seen that the solution of the present invention can achieve high and low regulation of the basal proliferation effect of T cells, thereby meeting the needs of verifying different functional drugs.
[0048] from Figure 3 It can be seen that the cell survival rate is maintained at around 30%.
[0049] Example 2
[0050] Using the method and culture medium formulation of Example 1, 96-well plates were coated with 50 μL of 2.5 μg / ml anti-CD3e antibody and cells were cultured in 200 μL of T-cell culture medium containing 27.5 μM β-mercaptoethanol to verify the immune-activating function of a test drug with unknown function. Figure 4 , Figure 5 It can be seen that, compared with the control group, the immune cell proliferation rate of the group with the added drug increased by more than 15%, indicating that the drug has an immune-promoting effect.
Claims
1. A method for in vitro culture and proliferation of T cells, characterized in that, CD3e antibody and β-mercaptoethanol were used to control the basal proliferation of T cells; Preferably, the mass ratio of the CD3e antibody to β-mercaptoethanol is 0.04-0.
8.
2. The method according to claim 1, characterized in that, Specifically, the following steps are included: (1) Antibody coating with CD3e antibody; (2) Prepare a single-cell suspension of lymphocytes and stain them; (3) Resuspend the cell suspension from step (2) in a culture medium containing β-mercaptoethanol; (4) After removing the solution in step (1), add the cell suspension from step (3), incubate, and then perform flow cytometry detection.
3. The method according to claim 2, characterized in that, The process of step (1) is as follows: the anti-CD3e antibody is incubated in a 37°C incubator for 2 hours to coat the antibody.
4. The method according to claim 2, characterized in that, The process of step (2) is as follows: S1. A single-cell suspension of lymphocytes was stained with CFSE, incubated at room temperature in the dark, and then added with complete culture medium to release unbound CFSE. The suspension was then incubated at room temperature in the dark and washed with complete culture medium. S2 cells were resuspended in a medium containing 15% fetal bovine serum, 1% penicillin-streptomycin, 20 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 5 ng / mL IL-2, and 13.75 μM–55 μM β-mercaptoethanol to a concentration of 3–5 × 10⁻⁶ cells / mL. 6 per mL.
5. The method according to claim 2, characterized in that, The process of step (3) is as follows: remove the anti-CD3e antibody solution coated in step (1), add the cell suspension from step (2), incubate in a 37°C incubator for three days, and then detect the cells using flow cytometry.
6. The method according to claim 5, characterized in that, The fetal bovine serum in step S1 needs to be inactivated by heat; and / or, The culture medium needs to be filtered.
7. The method according to claim 6, characterized in that, The conditions for heat inactivation are 50-60℃ for 10-50 minutes.
8. The application of the method according to any one of claims 1-7 in the in vitro culture of T cell proliferation; preferably, the application is for detecting the activity of immunomodulatory agents.
9. A kit for detecting the activity of an immunomodulator, characterized in that, It is prepared using the raw materials described in any one of claims 107.
10. The reagent kit according to claim 9, characterized in that, The immunomodulatory agents mentioned are immunosuppressants and / or immune activators.